Role of α-Subunit VISIT-DG Sequence Residues Ser-347 and Gly-351 in the Catalytic Sites of Escherichia coli ATP Synthase

This paper describes the role of alpha-subunit VISIT-DG sequence residues alpha Ser-347 and alpha Gly-351 in catalytic sites of Escherichia coli F1F0 ATP synthase. X-ray structures show the very highly conserved alpha-subunit VISIT-DG sequence in close proximity to the conserved phosphate-binding residues alpha Arg-376, beta Arg-182, beta Lys-155, and beta Arg-246 in the phosphate-binding subdomain. Mutations alpha S347Q and alpha G351Q caused loss of oxidative phosphorylation and reduced ATPase activity of F1F0 in membranes by 100- and 150-fold, respectively, whereas alpha S347A mutation showed only a 13-fold loss of activity and also retained some oxidative phosphorylation activity. The ATPase of alpha S347Q mutant was not inhibited, and the alpha S347A mutant was slightly inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium, in contrast to wild type and alpha G351Q mutant. Whereas 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) inhibited wild type and alpha G351Q mutant ATPase essentially completely, ATPase in alpha S347A or alpha S347Q mutant was inhibited maximally by similar to 80-90%, although reaction still occurred at residue beta Tyr-297, proximal to the alpha-subunit VISIT-DG sequence, near the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in beta E(empty) catalytic sites, as shown previously by x-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild type and alpha G351Q mutant but not in alpha S347Q or alpha S347A mutant. The results demonstrate that alpha Ser-347 is an additional residue involved in phosphate-binding and transition state stabilization in ATP synthase catalytic sites. In contrast, alpha Gly-351, although strongly conserved and clearly important for function, appears not to play a direct role.